Recently, a new family of IFN-gamma-induced p47 GTPases has been identified that is essential for innate immunity against intracellular pathogens. Knock-out (KO) mouse studies have shown that three of these proteins (LRG-47, IGTP, and IRG-47) are critical for resistance to multiple category A, B, and C biodefense priority pathogens. In particular, LRG-47, but not IGTP or IRG-47, is absolutely required for resistance to the intracellular bacteria Salmonella typhimurium, Mycobacterium tuberculosis, Listeria monocytogenes, and Francisella tularensis. In contrast, all three proteins are required for resistance to the protozoan parasite Toxoplasma gondii. The underlying mechanism for the antibacterial actions of LRG-47 is unknown. It is hypothesized here that LRG-47 regulates host resistance to intracellular bacteria by promoting IFN-gamma-induced bacterial killing in macrophages. It is further hypothesized that the protein localizes to the early endosomal compartment, where it catalyzes endosomal fusion with nascent bacteria-containing phagosomes, promoting phagosomal maturation and bacterial killing. In contrast, it is proposed that IGTP and IRG-47 localize to lysosomes and catalyze different facets of phagosomal processing. These hypotheses will be tested with the following aims: Aim I. The subcellular mechanism that underlies the impaired ability of LRG-47 KO macrophages to elicit IFN-gamma-induced bacterial killing will be defined, by determining: (a) the precise localization of LRG-47 within endosomal compartments; (b) the kinetics of LRG-47 trafficking to S. typhimurium-containing phagosomes; and (c) the effect of LRG-47 on maturation of the phagosome maturation, including lysosomal fusion, phagosomal acidification, and trafficking of endosomal markers. Aim II. Molecular domains of LRG-47 that determine its activity will be defined. Extensive mutational analysis and functional assays will define domains required for: (a) association with endosomes and trafficking to phagosomes; (b) endosome/phagosome fusion; (c) modulation of phagosomal maturation; and (d) bacterial killing in cultured macrophages and in vivo. Thus, this research will elucidate a mechanism that is critical for IFN-gamma- induced innate resistance to intracellular bacteria, with the ultimate goal of creating broadly effective anti-bacterial agents for biodefense.